Project Summary Aedes aegypti is the principal vector of Zika virus and several other medically-important arboviruses, such as chikungunya and dengue. Presently, efforts to control mosquito-borne diseases rely heavily on the use of insecticides targeting the nervous system (e.g., pyrethroids) to reduce mosquito populations. However, the emergence of insecticide resistance in mosquitoes is reducing the efficacy of these control agents. Thus, new insecticides with novel mechanisms of action are needed. We have previously demonstrated that 1) inward-rectifier potassium (Kir) channels perform fundamental roles in mosquito excretion and reproduction, and 2) small-molecule inhibitors of mosquito Kir channels elicit toxic effects on mosquitoes. Thus, Kir channels represent useful molecular targets for developing insecticides with novel mechanisms of action. The proposed work aims to develop small molecule inhibitors of Kir channels into resistance-breaking mosquitocides that would have nominal effects on humans and honey bees (Apis mellifera). Aim 1 will use leading-edge drug discovery approaches to develop analogs of 3 inhibitors of the Ae. aegypti Kir1 channel (AeKir1) that exhibit topical toxicity on mosquitoes. We will identify analogs that potently inhibit AeKir1, but do not inhibit a panel of human Kir channels and the honey bee Kir1 channel in vitro. Aim 2 will utilize in vivo toxicology assays in 1) pyrethroid-susceptible and pyrethroid-resistant adult female Ae. aegypti and 2) adult honey bees to evaluate the toxicity, resistance-breaking potential, and species- selectivity of the analogs. Moreover, the analogs will be tested in vitro with radioligand binding assays to assess their interactions with high-priority mammalian off-targets and thereby their potential human toxicity. Taken together, the results from the two aims will yield a diverse collection of potent and selective AeKir1 inhibitor analogs and identify those that exhibit the greatest potential for development into novel, safe mosquitocides for controlling the primary vector of Zika, dengue, and chikungunya viruses.